1. Technical Field
The present invention relates to methods of producing form-specific antibodies for transforming growth factor (TGF)-β subfamily compositions. More specifically, the present invention relates to methods of diagnosing cancer patients by measuring levels using antibodies.
2. Background Art
Nonsteroidal anti-inflammatory drug-activated gene (NAG-1)/macrophage inhibitory cytokine (MIC-1)/prostate-derived factor (PDF)-15/growth differentiation factor 15 (GDF)-15 is a member of TGF-β subfamily that plays an important role in pro-apoptotic and anti-tumoric activities as well as anti-inflammatory response to infection (1,2). High NAG-1 protein expression has been observed in tumors (3) and cardiovascular diseases (4) and became a therapeutic target.
Cleaved, matured NAG-1 expression increased in the serum of patients with the progression of the prostate cancer metastasis including bone metastasis (5,6). However, in a 2006 study with 462 control and 538 prostate cancer patients, it has been reported that serum NAG-1 levels were lower in the prostate cancer group compared with benign disease (BPH) and control (7).
The human NAG-1 protein is synthesized as a 308-amino acid pro-peptide, cleaved by a protease to a mature 112-amino acid protein and secreted as a disulfide-linked homodimer (8). A single-nucleotide polymorphism (SNP) at position 6 of the mature protein (code 604, CAC to GAC) results in a substitution of histidine to aspartic acid (H6D), which has a potential to alter the function of the protein (9). Indeed, a genotyping and sequencing analysis of genomic DNA for 1383 cases of prostate cancer and 789 control subjects in Sweden demonstrated that the H6D polymorphism in NAG-1/MIC-1 gene is associated with sporadic and familial cases of prostate cancer (9). A genotyping of genomic DNA for 819 cases of prostate cancer and 731 control subjects in Australia found that HD or DD genotype had increased risk of death from prostate cancer compared with HH genotype (10).
Compared to the DNA genotyping, serum genotyping is more convenient and faster because blood sampling is a routine at clinics and the ELISA result is obtained in 2-4 hours but requires antibodies specific for each wild type and mutated protein. Previously, a Mab-based genotyping assay has been developed to analyze the major allelic forms of NAG-1/MIC-1 on the basis of one of their monoclonal antibodies (13C4H4) which had a markedly reduced affinity for the MIC-1 D protein (4,11), because a H6D-specific antibody is not available. The genotype of a serum sample was determined by ratio of the 13C4H4 assay level to the level of total NAG-1 detected by 26G6H6 assay. The HH (homozygous H), HD (heterozygous) and DD (homozygous D) genotypes were determined by ratio ranges of 0.6-1.4, 0-0.45 and <0, respectively. The ranges of ratios, 0.6-1.4, 0-0.45 and <0, respectively, were randomly decided. Otherwise, it had to be 1, 0.5 and 0, respectively, or 0.75-1.25, 0.25-0.75 and 0-0.25, respectively. By looking at the ratios to predict the genotype, this method would not work because a negative ration would be needed to obtain a homozygous D genotype. The ELISA result obtained with a plate coated with the 13C4H4 monoclonal antibodies produced by immunization of His-containing wide type NAG-1 was inversely related with H6D protein concentration. Addition of 20 pg/ml and 70 pg/ml H6D proteins in the ELISA produced an optical density (OD) 20% and 30%, respectively, lower than the background OD (4,11).
Therefore, there remains a need to produce form-specific antibodies for wild type NAG-1 and HD6 that distinguish His from Asp at the 6th position of the proteins.